2-deoxy-isoguanosines isoteric analogues and isoguanosine containing oligonucleotides

ABSTRACT

2&#39;-Deoxyisoguanosine, isosteric analogues and isoguanosine derivatives of formulae I-V, processes for their production via compounds of the general formulae a or b and reaction with aroyl isocyanates or from compounds of the general formulae VI-IX by photochemical irradiation. A further production process is the conversion of deoxyguanosines or guanosines by means of persilylation, reaction with ammonia and deamination in the 2 position. The compounds are suitable as pharmaceutical agents with antiviral efficacy.

This application is a continuation of Ser. No. 08/257,724 filed Jun. 9,1994, abandoned, which is a 371 of PCT/EP/92/02843, filed Dec. 9, 1992.

Nucleosides are widespread in the living world as building blocks ofnucleic acids. They occur as ribonucleosides in ribonucleic acids (RNA)and as deoxyribonucleosides in deoxyribonucleic acids (DNA). Naturallyoccurring nucleosides are usually composed of a sugar moiety (ribose ordeoxyribose) and an aglyconic heterocyclic moiety. These so-callednucleobases are usually adenine, guanine, cytosine and thymine oruracil.

In addition nucleosides have been found in natural materials that arenot components of nucleic acids such as e.g. isoguanosine or1-methyl-isoguanosine. They often have interesting pharmacologicalproperties. The object of the present invention was to provide2'-deoxy-isoguanosines, isosteric derivatives and isoguanosinederivatives as well as their phosphorus compounds. Oligodeoxynucleotidesor DNA fragments which contain the compounds according to the inventionare suitable for inhibiting the expression of viral genes in biologicalsystems.

The invention concerns compounds of the general formulae I to IV (cf.FIGS. 1 and 2 for graphic formulae) in which

R₁ =hydrogen or a protecting group, PO₃ H₂, P₂ O₆ H₃, P₃ O₉ H₄ or thecorresponding alpha, beta and gamma thiophosphates provided that R₁ doesnot denote P₃ O₉ H₄ in formula III;

R₂ =hydrogen, hydroxy, phosphoramidite, methylphosphonate,H-phosphonate, a reporter or intercalator group;

R₃ =hydrogen or a protecting group;

W or/and Z=hydrogen, halogen, NH--(CH₂)_(n) NH₂ (n=2-12), R--CH₂ COOH(R=alkyl-C 1-8), a reporter or intercalator group.

The invention also concerns compounds of the general formula V in which

R₁ =equals hydrogen or a protecting group, PO₃ H₂, P₂ O₆ H₃, P₃ O₉ H₄ orthe corresponding alpha, beta or gamma thiophosphates;

R₂ =equals hydrogen, hydroxy, phosphoramidite, H-phosphonate,methylphosphonate, a reporter or intercalator group;

R₃ =hydrogen or a protecting group;

R₄ =hydroxy;

Z=hydrogen, halogen, NH--(CH₂)_(n) NH₂ (N=2-12), R--CH₂ --COOH (R=alkylwith C1-C8) a reporter or intercalator group

provided that R₁, R₃ and Z are not simultaneously hydrogen or R₁ is notsimultaneously P₂ O₆ H₃ when R₃ and Z are hydrogen.

The compounds according to the invention of formulae I-IV are producedby either starting with compounds of the general formulae a or b inwhich

R'=R" and represents an acyl protecting group such as e.g. p-toluoyl orbenzoyl, this is reacted with aroyl isocyanates e.g. benzoyl isocyanateand the desired isoguanosine is isolated,

or compounds of the general formulae VI to IX in which W, Z, R₁, R₂ andR₃ have the above-mentioned meanings,

and Hal represents chlorine, bromine or iodine

are converted photochemically by irradiation into the2'-deoxyisoguanosines.

Compounds of the general formula V are produced analogously in whichcase one starts with compounds of formulae a, b, VI to IX which containa β-D-ribo-furanosyl residue instead of the β-D-erythro-pentofuranosylresidue.

A particularly preferred process for the production of the compoundsaccording to the invention is to persilylate deoxyguanosine or guanosineand their derivatives with hexamethyldisilazane andtrimethyl-chlorosilazane. Subsequently the 2,6-diaminonucleoside isproduced using ammonia and tris(trimethyl)silyltriflate and selectivelydeaminated in the 2 position using nitrite to the isoguanosine ordeoxyguanosine.

The further derivatization is carried out according to methods wellknown to a person skilled in the art.

The heterocyclic moiety in the 2'-deoxyisoguanosines and isoguanosinesaccording to the invention can be preferably replaced by thecorresponding isosters 7-deaza-isoguanine and 7-deaza-8-aza-isoguaninein which case these bases can in addition contain further substituentson the C-7 of the 7-deaza- or 7-deaza-8-aza-isoguanine and on the C-8 ofisoguanine. Such substituents can for example be reporter groups asdescribed below.

Hydrogen, halogen, NH--(CH₂)_(n) NH₂, R--CH₂ COOH, a reporter orintercalator group are particularly preferred.

A diamino group can be introduced at position W and/or Z by halogenatinga compound in which W and/or Z is hydrogen (for example with brominewater) and introducing a diamino group in the bromide by nucleophilicsubstitution. A diaminopentyl or diamino-hexyl group is particularlypreferably introduced. The desired compound can be prepared from themixture of amino compounds by chromatographic purification.

A carboxyl group can be introduced by reacting the correspondinghalogenated compound with methyllithium and preparing methyl bromidewith halogen. This compound is reacted with aminocarboxylic acids (e.g.amino-caproic acid) to form the final product.

The reporter or intercalator groups are preferably coupled in theiractivated form (e.g. hydroxysuccinimide ester) to the amino or carboxylgroup of the compounds according to the invention.

All suitable end groups known to a person skilled in the art can bepresent on the 3' and 5' end of the sugar moiety of the compoundsaccording to the invention. Hydrogen, monophosphate, diphosphate ortriphosphate, a reporter group or intercalator group are preferred forthe 3' end and for the 5' end. A reporter group within the meaning ofthe invention is understood as a hapten such as biotin or digoxigenin ora fluorescent dye. Suitable intercalator groups are described by Helene,C. in "Antisense RNA and DNA, Curr. Commun. Mol. Biol.; Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y., 1987" and are preferablyphenanthroline, acridine, actinomycin or its chromophore or heavy metalcomplexing agents such as EDTA. Groups which result in a cross-linkingof nucleic acids such as e.g. psoralen are also advantageous.

Cyclic phosphoric acid diesters (3',5'-cyclophosphates) can be producedfrom the compounds according to the invention by water liberationbetween the 3'-OH and 5'-OH of the sugar moiety.

The production of phosphoramidites, H-phosphonates andp-methylphosphoramidites from isoguanosines is carried out analogouslyto the production of the corresponding deoxyisoguanosine derivatives inwhich case the 2'-OH group is preferably protected by atriisopropylsilyl group.

The invention in addition concerns 2'-deoxyisoguanosines, isoguanosinesand 7-deaza-8-aza-isoguanosine-3'-phosphoramidites, -3'-H-phosphonatesand -P-methyl-phosphoramidites protected by bases and sugars. Thesecompounds are suitable as nucleotide building blocks for the productionof oligonucleotides.

The nucleotide building blocks according to the invention preferablycontain protecting groups on the heterocyclic bases as well as on the5'-OH and/or 2'-OH groups of ribose.

Amino-protecting groups such as e.g. benzoyl, formamidine, isobutyryl ordiphenoxyacetyl groups are preferably used as a protecting group on theheterocyclic bases.

The 5' or 2'-OH protecting group of the sugar moiety is preferably atriphenylmethyl, monomethoxytrityl, dimethoxytrityl,t-butyl-dimethylsilyl, t-butyldiphenylsilyl, t-butyl-methoxyphenylsilylor pixyl group

3'-O-(2-cyanoethyl)-N,N-diisopropyl-aminophosphanes and3'-O-methyl-N,N-diisopropylamino-phosphanes are preferred asphosphoramidites. The H-phosphonates are preferably used as salts.

The production of the monomeric nucleotide building blocks is carriedout according to methods familiar to a person skilled in the art such asthose described by Gait, M. J. in "Oligonucleotide Synthesis, APractical Approach", IRL Press, Ltd. (1984).

The compounds according to the invention of the general formulae I to IXcan be incorporated by DNA or RNA polymerases into oligonucleotides ornewly synthesized DNA or RNA in the form of their respective5'-triphosphates or α-, β- or γ-thiotriphosphates.

In a particularly preferred embodiment, these nucleotide building blocksaccording to the invention are labelled with ³² P or ³⁵ S.

The invention therefore also concerns a process for the production ofoligonucleotides and polynucleotides which contain the compoundsaccording to the invention as building blocks. Such processes can be ofa chemical as well as of an enzymatic nature.

The chemical synthesis of oligonucleotides is carried out according tomethods known to a person skilled in the art as described for example inGait, M. J., loc. cit. or in Narang, S. A., "Synthesis and Applicationof DNA and RNA", Academic Press, 1987.

The production of the oligonucleotides according to the invention iscarried out in a known manner, for example according to the phosphatetriester, the phosphite triester or H-phosphonate method in ahomogeneous phase or on a support. The two latter methods are preferablyused whereby the synthesis is usually carried out using automatedsynthesizers. The support materials in this case consists of inorganic(controlled pore glass, Fractosil) or organic polymeric material (e.g.polystyrene) known to a person skilled in the art.

The invention therefore also concerns a process for the production ofoligonucleotides consisting of 6 to 100 nucleotide building blocksaccording to the oligonucleotide synthesis process in which, dependingon the sequence design, appropriate 2'-deoxy-isoguanosines orisoguanosines according to the invention are used wherein a startingnucleoside is bound to a solid support and subsequently the desiredoligonucleotide is assembled by stepwise coupling using appropriatelyactivated monomeric nucleotide building blocks, if desired trivalentphosphorus is oxidized to pentavalent phosphorus during or after thesynthesis, the oligonucleotide is cleaved from the support with a firstbase, heterocyclic protecting groups are cleaved with a second base, the5' protecting group is cleaved with an acid and the oligonucleotide ispurified if desired. The purification is preferably carried out byreverse phase or anion exchange HPLC. This is usually followed by adesalting, for example by dialysis.

In addition the oligonucleotides according to the invention can also beproduced enzymatically using polymerases. Such processes are known to aperson skilled in the art under the terms "in vitro transcription","nick translation" [Rigby et al., J. Mol. Biol. 113, 237 (1977)] and"random priming" [Feinberg, A. P. and Vogelstein, B., Anal. Biochem.137, 266 (1984)].

In this process 2'-deoxynucleoside-5'-triphosphates orisoguanosine-5'-triphosphates are basically incorporated by polymerasesusing a single-stranded template nucleic acid and a starter molecule(primer/promoter) into a newly synthesized second strand that iscomplementary to the bases of the first strand. The use of thenucleoside-5'-triphosphates according to the invention and theircorresponding substituted derivatives enables suitable signal groups orreporter groups such as haptens or fluorophores to be incorporated forexample into nucleic acids. Such techniques are widely used nowadays forexample in the form of non-radioactive labelling of biomolecules.

The invention in addition concerns the use of oligonucleotides in whichguanosine or deoxyguanosine building blocks have been replacedcompletely or partially by the deoxyisoguanosine or isoguanosinebuilding blocks according to the invention and which are composed of 6to 100 nucleotide building blocks for the production of a pharmaceuticalagent with antiviral activity.

Oligonucleotides containing 2'-deoxyisoguanosine form duplex and triplexstructures and also aggregates with themselves as well as with otherconventional and modified oligonucleotides. In the case of2'-deoxyisoguanosine this results in manifold base pairing patternswhich differ from those of 2'-deoxyguanosine and in which theoligonucleotide strands can be arranged either in parallel orantiparallel.

a) Aggregate structures

During the preparation of 2'-deoxyisoguanosine it is noticed that itforms gels like 2'-deoxyguanosine and crystallizes with extremedifficulty. Aggregates have been demonstrated by gel electrophoresis foroligonucleotides containing guanine [66] which comply with thestructural proposal of Zimmerman [67].

In this structure all N⁷ atoms are bound up in a Hoogsteen base pairing.The (iG_(d))₄ structure differs considerably from the (G_(d))₄configuration since in this case two N⁷ atoms and two N³ atoms act inaddition to the O atoms as proton acceptors. As a result2'-deoxyisoguanosine is able to form a dimeric structure in which onemolecule is present as a 1H tautomer and the other as a 3H tautomer.

An interaction via N-7 is not possible in the case of 7-deazapurinederivatives such as 7-deaza-2'-deoxyisoguanosine and7-deaza-8-aza-2'-deoxyisoguanosine. Hence no aggregate formation isobserved with these compounds.

b) Watson-Crick duplex structures

With regard to the complexation of single-stranded DNA or RNA,oligonucleotides containing iG_(d) are able to form duplex structures.In this process parallel and antiparallel configurations of the strandsare possible. Due to the altered pattern of substituents on the base andthe possible parallel duplex structures, an increased stability towardsnucleases would be expected.

c) Triplex structures of DNA duplexes with oligonucleotides containingiG_(d)

Oligonucleotides which contain 2'-deoxyisoguanosine as monomericbuilding blocks can form triplex structures with d(AT) duplexes as wellas with d(GC) duplexes. In both cases these structures can form in aneutral medium; a protonation is not necessary. As a result thecomplexation can be achieved under physiological conditions.

The structures discussed above apply in principle tooligoribonucleotides and oligodeoxyribonucleotides.

[66] J. Kim, Ch. Cheong and P. B. Moore, `Tetramerization of an RNAoligonucleotide containing a GGGG sequence`, Nature 1991, 351, 331.

[67] S. B. Zimmerman, `X-ray Study by Fiber Diffraction Methods of aSelf-aggregate of guanosine-5'-phosphates with the same HelicalParameters as Poly(rG)`, J. Mol. Biol. 1976, 106, 663.

The oligonucleotides according to the invention and their salts can beused as medicines e.g. in the form of pharmaceutical preparations whichcan be administered orally e.g. in the form of tablets, coated tablets,hard or soft gelatin capsules, solutions, emulsions or suspensions. Theycan also be administered rectally e.g. in the form of suppositories orparenterally e.g. in the form of injection solutions. These compoundscan be processed in therapeutically inert organic and inorganic carriersfor the production of pharmaceutical preparations. Examples of suchcarriers for tablets, coated tablets and hard gelatin capsules arelactose, maize starch or derivatives thereof, talcum, stearic acid orsalts thereof. Suitable carriers for the production of solutions arewater, polyols, sucrose, inverted sugar and glucose. Suitable carriersfor injection solutions are water, alcohols, polyols, glycerol andvegetable oils. Suitable carriers for suppositories are vegetable andhardened oils, waxes, fats and semi-liquid polyols.

The pharmaceutical preparations can also contain preservatives,solvents, stabilizers, wetting agents, emulsifiers, sweeteners, dyes,flavourings, salts to change the osmotic pressure, buffers, coatingagents or antioxidants as well as if desired othertherapeutically-active substances.

The invention is elucidated in more detail by the following examples:

EXAMPLE 1 2'-Deoxy-isoguanosine[9-(2'-deoxy-β-D-erythro-pentofuranosyl)-9H-isoguanine

Method A: 0.6 ml (4.3 mmol) benzoyl isocyanate is added at roomtemperature while stirring to a solution of 400 mg (0.87 mmol)5-amino-1-[2'-deoxy-3',5'-di-O-(p-toluoyl)-β-D-erythro-pentofuranosyl)-4-imidazocarbonitrilein 15 ml dry acetonitrile. A precipitate forms within 15 minutes. Thereaction mixture is stirred overnight and afterwards refluxed for 30minutes. The solvent is removed by distillation, the residue isdissolved in 100 ml of a mixture of isopropanol/25% aqueous ammoniasolution (1:1) and the solution is stirred for 2 days at roomtemperature. After evaporation, the residue is extracted twice with 50ml ether each time while stirring. The residue is taken up in 100 mlwater to which a few drops of 25% ammonia solution have been added andfiltered. The filtrate is concentrated to a volume of about 30 ml andapplied to a XAD-4 column (20×2 cm). The inorganic salts are removed byelution with 200 ml water, elution with water/isopropanol (9:1) affordsthe desired product. After evaporation, the residue is crystallized fromethanol.

Yield: 100 mg=43% of theoretical yield.

Colourless crystals which melt above 230° C. with decomposition.

TLC (cellulose, mobile solvent water-saturated n-butanol): R_(f) =0.25

UV (pH 1): λ_(max) 235 nm (5200), 284 (11500); (pH 7): 247 (9100), 292(10100) (pH 13): 249 (6600), 284 (9800).

¹ H-NMR (d₆ DMSO): 2.18, 2.30 (2 m, 2H, C-2'); 3.55 (m, 2H, C-5'); 3.84(m, 1H, C-4'); 4.35 (m, 1H, C-3'); 5.30 (bs, 2H, OH--C-3' and OH--C-5');6.11 (t, J=6.4 Hz, 1H, C-1'); 7.95 (s, 1H, C-8); 8.1 (bs, NH₂).

Elemental analysis C₁₀ H₁₃ N₅ O₄ : calc. C, 44.94; H, 4.90; N, 26.21;found C, 44.70; H, 5.03; N, 25.99.

Method B: A solution of 200 mg (0.61 mmol) 2-bromo-2'-deoxyadenosine in250 ml water is irradiated for 30 minutes in a quartz reactor (equippedwith a 30 W sterilisation lamp Philips/Holland; the radiation is passedthrough a 2 mm layer of 20% acetic acid in order to filter out lightbelow 230 nm). The pH is then adjusted to 8.0 with ammonia solution, itis concentrated to a volume of ca. 20 ml and applied to an XAD-4 column(21×3 cm). The elution is carried out with a gradient of water to 50%methanol whereby the desired product is eluted at 15-20% methanol. Thepooled main fraction is evaporated and the residue is crystallized fromethanol.

Yield: 85 mg=53% of theoretical yield.

The material is chromatographically identical to that obtained in methodA and with regard to its spectral data.

2'-Deoxy-isoguanosine is obtained in 52% yield in an analogous mannerfrom 2-chloro-2'-deoxy-adenosine after an irradiation period of 1 hour.

EXAMPLE 2 7-(2'-Deoxy-β-D-erythro-pentofuranosyl)-7H-isoguanine

0.27 ml (1.95 mmol) benzoyl isocyanate is added at room temperaturewhile stirring to a solution of 300 mg (0.65 mmol)4-amino-1-(2'-deoxy-3',5'-di-O-(4-toluoyl)-β-D-erythro-pentofuranosyl)-1H-imidazole-5-carbonitrilein 15 ml absolute acetonitrile. A white precipitate forms after 1 hour;the suspension is stirred overnight, evaporated to dryness andisopropanol/25% aqueous ammonia solution (75 ml, 1:1) is added. Afterstirring for 2 days at room temperature, the solvent is removed byevaporation, the residue is taken up in 75 ml diethylether/ethyl acetate(1:1) and stirred for 1 hour at room temperature. The supernatantsolution is decanted off and the residue is admixed with 75 ml water aswell as 2 drops of 25% aqueous ammonia solution. After stirring for ashort time, it is filtered, the filtrate is concentrated to 40 ml andapplied to an Amberlite XAD-4 column (25×3 cm). After washing with 200ml water, the main product is eluted with 10% aqueous isopropanol andsubsequently the solvent is removed by evaporation. It is crystallizedfrom ethanol.

Yield: 110 mg=63% of theoretical yield.

Colourless crystals which melt above 230° C. with decomposition.

TLC (cellulose, mobile solvent water-saturated n-butanol): R_(f) =0.15

UV (pH 1): λ_(max) 290 (8600); (pH 7): 282 (7100), 242 (7000); (pH 13):285 (5700).

¹ H-NMR (d₆ DMSO): 2.25, 2.40 (2 m, 2H, C-2'); 3.55 (m, 1H, C-5'); 3.84(m, 1H, C-4'); 5.35 (bs, 3'-OH, 5'-OH); 6.12 (pt, J=6.6 Hz, 1H, C-1');7.27 (bs, 2H, NH₂); 8.19 (s, 1H, C-8).

Elemental analysis C₁₀ H₁₃ N₅ O₄ : calc. C, 44.94; H, 4.90; N, 26.2;found C, 44.78; H, 5.05; N, 25.82.

EXAMPLE 3 7-Deaza-2'-deoxy-isoguanosine-5'-triphosphate[4-amino-7-(2'-deoxy-β-D-erythro-pentofuranosyl)-3H,7H-pyrrolo[2,3-d]-pyrimidin-2-one-5'-triphosphate]

Step 1: 7-deaza-2'-deoxy-isoguanosine

A solution of 85 mg (0.3 mmol)4-amino-2-chloro-7-(2'-deoxy-β-D-erythro-pentofuranosyl)-7H-pyrrolo[2,3-d]-pyrimidineis dissolved in 200 ml water containing 1 ml concentrated aqueousammonia solution and the solution is irradiated for 1 hour in a quartzreactor as described in example 1, method B. The solution issubsequently concentrated to 30 ml and applied to a XAD-4 column (20×2cm). The resin is subsequently washed with 150 ml water, afterwards itis eluted with ca. 1 l of a mixture of water/isopropanol 9:1. Thefractions containing the nucleoside are pooled and evaporated. Theresidue is crystallized from ethanol.

Yield: 46 mg=58% of theoretical yield.

Colourless needles of melting point 230° C.

Elemental analysis C₁₁ H₁₄ N₄ O₄ : calc. C, 49.62; H, 5.30; N, 21.04;found C, 49.7; H, 5.4; N, 21.1.

Step 2: 7-deaza-2'-deoxy-isoguanosine-5'-triphosphate

The nucleoside from step 1 was converted into the 5'-monophosphateaccording to Yoshikawa et al. [Tetrah. Lett. 50, 5065 (1967)]; thedesired triphosphate was obtained therefrom according to the method ofHoard and Ott [J. Am. Chem. Soc. 87, 1785 (1965)].

³¹ P-NMR (0.1 M EDTA/D₂ O/Eth₃ N): -8.20 (d, P-g), -10.5 (d, P-a), -21.2(t, P-β)

EXAMPLE 44-Amino-6-chloro-1-(2'-deoxy-β-D-erythropentofuranosyl)-1H-pyrazolo[3,4]-pyrimidine

A suspension of 540 mg (1 mmol)1-{2'-deoxy-3',5'-di-O-(4-toluoyl)-β-D-erythro-pentofuranosyl}-4,6-dichloro-1H-pyrazolo[3,4-d]-pyrimidinein 40 ml methanolic ammonia solution (saturated at 0° C.) is stirred for2 days at 60° C. The reaction mixture is then evaporated to dryness andthe residue is chromatographed on a silica gel 60 H column (15×4 cm)using CH₂ Cl₂ /methanol (9:1).

Yield: 200 mg=70% of theoretical yield

Recrystallization from water yields colourless needles of melting point183-185° C.

TLC (silica gel, mobile solvent CH₂ Cl₂ /methanol 9:1): R_(f) =0.45.

UV (pH 1): λ_(max) =265 (10150); (pH 7): 271.5 (10350).

¹ H-NMR (d₆ DMSO): 2.45, 2.75 (2 m, 2H, C-2'); 3.50 (m, 2H, C-5'); 3.81(q, 1H, C-4'); 4.41 (bs, 1H, C-3'); 4.71 (t, OH--C-5'); 5.27 (d,OH--C-3'); 6.44 (t, J=6.2 Hz, 1H, C-1'); 8.16 (bs, NH₂); 8.34 (s, 1H,C-3).

Elemental analysis C₁₀ H₁₂ N₅ O₃ Cl: calc. C, 42.04; H, 4.23; N, 24.51;found C, 42.29; H, 4.27; N, 24.48.

EXAMPLE 54-Amino-1-(2'-deoxy-β-D-erythro-pentofuranosyl)-1H-pyrazolo[3,4-d]pyrimidin-6(5H)-one

A solution of 105 mg (0.37 mmol)4-amino-6-chloro-1-(2'-deoxy-β-D-erythro-pentofuranosyl)-1H-pyrazolo[3,4-d]pyrimidineis dissolved in 200 ml water containing 1 ml concentrated ammoniasolution and irradiated for 1 hour in a quartz reactor. The processingis carried out as described in example 3.

Yield: 57 mg=58% of theoretical yield.

Colourless powder, melting point 235° C. (decomp.)

TLC (cellulose, water-saturated n-butanol): R_(f) 0.3

UV (pH 1): 268 (8500); (pH 7): 221 (21200), 251 (7900), 283 (6700); (pH13): 221 (26600), 255 (6600), 278 (9000).

¹ H-NMR (d₆ DMSO): 2.20, 2.52 (2 m, 2H--C-2'); 3.3 (m, 2H, C-5'); 3.64(q, H, C-4'); 4.21 (bs, H, C-3'); 5.1 (bs, OH--C-3' and OH--C-5'); 6.12(t, J=6.4, H--C-1'); 7.79 (s, H--C-3); 8.1 (NH₂).

Elemental analysis C₁₀ H₁₃ N₅ O₄ : calc. C, 44.94; H, 4.90; N, 26.2;found C, 44.89; H, 5.0; N, 25.89.

EXAMPLE 6 9-(2'-deoxy-β-D-erythro-pentofuranosyl)-6-{[1-(dimethylamino)methylene]-9H-isoguanine (5)

200 mg (0.74 mmol) 2'-deoxy-isoguanosine from example 1 is dissolved in10 ml absolute dimethylformamide and admixed with 1.3 ml (7.6 mmol)N,N-dimethylformamide-diethylacetal. It is stirred for 24 hours at roomtemperature, concentrated in a vacuum and the residue is evaporatedseveral times with toluene and acetone. Subsequently it ischromatographed on silica gel 60 H (25×4.5 cm column, eluting agentchloroform/methanol 8:2). The main fractions are pooled, the solvent isremoved by evaporation and the residue is recrystallized from methanol.

Yield: 150 mg=62% of theoretical yield

Pale yellow crystals of melting point 178° C. (decomp.).

TLC (silica gel, chloroform/methanol 8:2) R_(f) =0.62

UV (methanol: λ_(max) =338 (9400), 259 (6100), 223 (9100).

¹ H-NMR (d₆ DMSO): 2.20, 2.64 (2 m, 2H, C-2'); 3.12, 3.22 (2 s, 4H,(CH₃)₂ N); 3.50 (2 m, 2H, C-5'); 3.86 (m, 1H, C-4'); 4.38 (m, 1H, C-3');5.10 (t, 1H, OH--C-5'); 5.31 (d, 1H, OH--C-3'); 6.15 (pt, J=6.25 Hz, 1H,C-1'); 8.08 (s, 1H, C-8); 9.2 (s, 1H, --N═CH).

Elemental analysis: C₁₃ H₁₈ N₆ O₄ : calc. C, 48.44; H, 5.63; N, 26.07;found C, 48.25; H, 5.69; N, 25.87.

EXAMPLE 77-(2'-deoxy-β-D-erythro-pentofuranosyl)-6-[1-(dimethylamino)methylidene]-7H-isoguanine

300 mg (1.12 mmol) 7-(2'-deoxy-β-D-erythro-pentofuranosyl)-7H-isoguanineis dissolved in 10 ml absolute dimethylformamide and admixed with 2 ml(11.7 mmol) N,N-dimethylformamide-diethylacetal and stirred for 24 hoursat room temperature. The processing is carried out as described inexample 6.

Yield: 62 mg=72% of theoretical yield.

Pale yellow crystals of melting point 208-209° C.:

TLC (silica gel, chloroform/methanol 8:2): R_(f) =0.56

UV (methanol) λ_(max) =326 (7800), 296 (6700), 245 (3200).

¹ H-NMR (d₆ DMSO): 2.30 2.43 (2 m, 2H, C-2'); 3.14, 3.23 (2 s, 4H,(CH₃)₂ N); 3.49 (2 m, 2H, C-5'); 3.9 (m, 1H, C-4'); 4.30 (m, 1H, C-3');5.10 (t, 1H, OH--C-5'); 5.31 (d, 1H, OH--C-3'); 6.84 (pt, J=6.25 Hz, 1H,C-1'); 8.32 (s, 1H, C-8); 8.8 (s, 1H, --N═CH).

Elemental analysis: C₁₃ H₁₈ N₆ O₄ : calc. C, 48.44; H, 5.63; N, 26.07;found C, 48.29; H, 5.73; N, 25.91

EXAMPLE 89-(2-deoxy-β-D-erythro-pentofuranosyl)-6-[1-(dimethylamino)methylidene]-2-chloro-adenosine

300 mg (1.05 mmol) 2-chloro-2'-deoxy-adenosine [produced according to Z.Kazimierczuk et al., J. Am. Chem. Soc. 106, 6379 (1984)] is dissolved in3 ml anhydrous DMF and 0.9 ml (5.2 mmol)N,N-dimethylformamide-diethylacetal is added. It is stirred for 1 hourat room temperature, concentrated and the residue is evaporated severaltimes with toluene. Subsequently it is chromatographed on silica gel 60H (15×5 cm column, eluting agent dichloromethane/methanol 9:1).

Yield: 295 mg=82.4% of theoretical yield.

Colourless, foamy product.

The 7-regioisomers can be synthesized in an analogous manner accordingto Z. Kazimierczuk.

EXAMPLE 99-(2'-deoxy-β-D-erythro-pentofuranosyl)-5'-O-(4,4-dimethoxytriphenyl-methyl)-6-[1-(dimethylamino)methylidene]2-chloro-adenosine

340 mg (1 mmol) of the compound from example 8 is dried by evaporatingseveral times with anhydrous pyridine and subsequently dissolved in 2 mlpyridine. After addition of 440 mg (1.3 mmol)4,4'-dimethoxytriphenylmethyl chloride, it is stirred for 1 hour at roomtemperature. Afterwards it is hydrolysed with 30 ml of a 5% aqueousNaHCO₃ solution and extracted three times with 50 ml dichloromethaneeach time. The combined organic phases are dried over Na₂ SO₄, filteredand the solvent is removed. After chromatography on silica gel 60 H(20×5 cm column, mobile solvent dichloromethane/methanol 9:1) andevaporating the main fraction, the product is obtained as an amorphousmaterial.

Yield: 390 mg=60% of theoretical yield.

TLC (silica gel, mobile solvent dichloromethane/methanol 9:1): R_(f)=0.41

UV (methanol): λ_(max) =314 (27200), 236 (30500).

¹ H-NMR (d₆ DMSO): 2.37, 2.82 (2 m, 2H, C-2'); 3.14, 3.22 (2 s, 6H,(CH₃)₂ N); 3.69, 3.71 (2 s, 6H, 2×O--CH³); 3.98 (m, 1H, C-4'); 4.46 (m,1H, C-3'); 5.39 (d, J=4.5 Hz, 1H, OH--C-3'); 6.35 (pt, J=6.2 Hz, 1H,C-1'); 6.72-7.3 (m, 13H, arom.); 8.37 (s, 1H, C-8); 8.84 (s, 1H,--N═CH).

Elemental analysis C₃₄ H₃₅ N₆ O₅ Cl: calc. C, 63.49; H, 5.49; N, 13.06;found C, 63.30; H, 5.68; N, 12.88.

EXAMPLE 109-(2'-deoxy-β-D-erythro-pentofuranosyl)-3'-(H-phosphonato)-5'-O-(4,4-di-methoxytriphenylmethyl)-6-[1-(dimethylamino)methylidene]-2-chloro-adenosine

358 mg (5 mmol) 1,2,4-triazole is added under an argon atmosphere atroom temperature to a solution of 12 ml absolute CH₂ Cl₂, 135 μl (1.55mmol) PCl₃ and 1.7 ml (15.2 mmol) N-methylmorpholine. After stirring for30 minutes, the reaction solution is cooled to 0° C. and admixed within10 minutes with a solution of 200 mg (0.31 mmol) of the compound fromexample 9 in 10 ml CH₂ Cl₂. It is stirred for a further 20 minutes atroom temperature and subsequently 17 ml 1 M triethylammonium bicarbonatebuffer (TBK) (pH 7.7) is added. The aqueous phase is extracted threetimes with 15 ml CH₂ Cl₂ in each case, the combined organic phases aredried over Na₂ SO₄ and the dichloromethane is removed by evaporation.After chromatography on silica gel 60 H (20×5 cm column, elution firstlywith 1 l dichloromethane/triethylamine 98:2, then withdichloromethane/methanol/triethylamine 88:10:2), the main fractions arepooled, the solvent is removed, the residue is taken up in 20 ml CH₂ Cl₂and shaken out several times with 0.1 M TBK buffer. The combined organicphases are dried over Na₂ SO₄ and concentrated.

Yield: 190 mg=73% of theoretical yield.

TLC (silica gel, mobile solvent dichloromethane/methanol/triethylamine88:10:2): R_(f) =0.37

UV (methanol): λ_(max) =316 (19900), 235 (21200).

¹ H-NMR (d₆ DMSO): 0.9-1.2 (m, 9H, HN(CH₂ CH₃)₃); 2.92 (2 m, 2H, C-2');2.60-2.64 (m, 6H, NH(CH₂ CH₃)₃); 3.14, 3.23 (2 s, 6H, (CH₃)₂ N9; 3.69,3.71 (2 s, 6H, 2×OCH₃); 4.16 (m, 1H, C-4'); 4.77 (m, 1H, C-3'); 6.63 (d,1H, H-phosphonate); 6.34 (pt, 1H, C-1'); 6.73-7.3 (m, 13H, arom.); 8.34(s, 1H, C-8); 8.85 (s, 1H, --N═CH).

³¹ P-NMR (d₆ DMSO): 1.09 ppm (¹ J (P-H)=585.3 Hz, ³ J (P-H)=9.1 Hz).Elemental analysis C₄₀ H₅₁ N₇ O₇ ClP: calc. C, 59.43; H, 6.36; N, 12.13;found C, 59.26; H, 6.50; N, 11.93.

EXAMPLE 119-(2'-deoxy-β-D-erythro-pentofuranosyl)-5'-O-(4,4'-dimethoxytriphenyl-methyl)-6-[1-dimethylamino)-methylidene]-9H-isoguanine

322 mg (1 mmol) of the compound from example 6 is converted into thedesired 5'-dimethoxytrityl-protected nucleoside as described in example9.

Yield: 355 mg=57% of theoretical yield.

TLC (silica gel, dichloromethane/methanol 9:1): R_(f) =0.32.

¹ H-NMR (d₆ DMSO): 2.35, 2.7 (2m, 2H, C-2'); 3.14, 3.24 (2 s, 6H, (CH₃)₂N); 3.61, 3.69 (2 s, 6H, 2×OCH₃); 4.10 (m, 1H, C-4'); 4.41 (m, 1H,C-3'); 5.28 (d, J=4.5 Hz, 1H, OH--C-3'); 6.40 (pt, J=6.2 Hz, 1H, C-1');6.68-7.2 (m, 13H, arom.); 8.4 (s, 1H, C-8); 8.8 (s, 1H, --N═CH).

Elemental analysis C₃₄ H₃₆ N₆ O₆ : calc. C, 65.37; H, 5.81; N, 13.45;found C, 65.23; H, 5.92; N, 13.18.

EXAMPLE 12 Carrier(Fractosil)-bound9-[2'-deoxy-β-D-erythro-pentofuranosyl-5'-O-(4,4'-dimethxytriphenylmethyl)]-6-[(1-dimethyl-amino)methylidene]-9H-isoguanine

75 mg (0.6 mmol) N,N-dimethylamino-pyridine and 250 mg (2.5 mmol)succinic acid anhydride are added to a solution of 312.3 mg (0.5 mmol)of the completely protected nucleoside from example 11 in 15 ml drypyridine and the reaction mixture is stirred for 72 hours at 40° C.After addition of 5 ml water, it is evaporated to dryness and theresidue is rotary evaporated with 50 ml toluene. It is dissolved in 100ml dichloromethane and extracted in succession firstly with 30 ml 10%aqueous citric acid solution and then with 30 ml water. After drying theorganic phase over Na₂ SO₄, the solvent is removed by distillation. 335mg (nearly the theoretical yield) of the succinate is obtained which isreacted without further purification. For this it is dissolved in 2.5 mlof a 5% solution of pyridine in dioxane and admixed with 120 mg (0.9mmol) p-nitrophenol and 206 mg (1 mmol) dicyclohexyl-carbodiimide. It isstirred for 3 hours at room temperature, the precipitateddicyclohexylurea is removed by suction filtration and 3 ml DMF, 600 mgFractosil® 200 (Merck, 450 μeq NH₂ /g) and 0.625 ml triethylamine areadded to the filtrate. It is shaken for 4 hours at room temperature,then 0.2 ml acetic anhydride is added and it is shaken for a further 30minutes. The silica gel containing the bound nucleoside is suctionfiltered, washed in succession with DMF, ethanol and ether, and dried ina vacuum.

The loading of the carrier with nucleoside was determined as follows:

5 mg Fractosil carrier was treated with 10 ml 0.1 m p-toluenesulfonicacid in acetonitrile. After standing for 10 minutes, the supernatant wasmeasured spectrophotometrically at 498 nm in which case a loading of 35μmol nucleoside per gram carrier was found (A_(DMT) =70,000).

EXAMPLE 139-(2'-deoxy-β-D-erythro-pentofuranosyl)-5'-O-(4,4-'dimethoxy-triphenylmethyl)-6-[1-dimethylamino)-methylene]-9H-isoguanine-3'-(2-cyanoethyl)-N,N-diisopropyl-phosphoramidite

312.3 mg (0.5 mmol) of the protected nucleoside from example 11 isdissolved in 5 ml dry tetrahydrofuran and 0.275 ml (1.6 mmol)ethyldiisopropylamine is added. Subsequently 0.125 ml (0.55 mmol)chloro-β-cyanoethoxy-(N,N-diisopropylamino)-phosphane is added dropwisewithin ca. 3 minutes under N₂. The reaction mixture is stirred for 30minutes at room temperature, then ca. 10 ml aqueous 5% NaHCO₃ solutionis added and afterwards it is extracted twice with ca. 10 mldichloromethane each time. The combined organic phases are dried overNa₂ SO₄, the solvent is removed by distillation and the residue ischromatographed on silica gel 60 H using the mobile solvent CH₂ Cl₂/ethyl acetate/triethylamine, 45:45:10.

Yield: 380 mg=94% of theoretical yield.

TLC (silica gel, mobile solvent dichloromethane/ethylacetate/triethylamine 45:45:10): R_(f) =0.42 and 0.45 (2 diastereomers).

³¹ P-NMR (d₆ DMSO): 147 and 152 ppm (2 diastereomers).

EXAMPLE 14

Solid phase synthesis of the oligonucleotide d(AAA^(9i) G) viaphosphoramidite

The synthesis of the oligonucleotide was carried out on a 1 μmol scaleusing the commercially available 5'-O-(4,4'-dimethoxy-trityl)-N⁶-benzoyl-2'-deoxy-adenosine-3'-[(2-cyanoethoxy)-N,N-di-isopropylamino]phosphaneand the phosphoramidite obtained in example 12 according to the standardprotocol for the DNA-synthesizer 380 B from the Applied BiosystemsCompany (User Manual). The cleavage of the 5'-tritylated oligomer fromthe carrier material is carried out at room temperature in 25% aqueousammonia solution.

The protecting groups of the exocyclic amino groups on the heterocyclewere removed by a 16 hour treatment with 25% aqueous ammonia solution at60° C. The 4,4'-dimethoxytrityl protecting group of the oligomer wascleaved by treatment with 80% acetic acid during 5 minutes at roomtemperature.

The oligonucleotide was purified by means of HPLC on a RP-18 columnusing the following mobile solvent system:

Mobile solvent A: 5% acetonitrile in 0.1 M triethylammonium acetatebuffer, pH 7.0

Mobile solvent B: 100% acetonitrile

Gradient 25 minutes 0-20% B in A

It was desalted on a RP-18 silica gel column by firstly removinginorganic material by washing with water and then the oligomer waseluted with a mixture of methanol/water (3:2).

Yield: 5.5 A₂₆₀ units=ca. 50% of theoretical yield.

EXAMPLE 15

Chemical solid phase synthesis of the oligonucleotide d(AAAA^(Cl) A) viaphosphonate

The synthesis of this oligonucleotide with a 2-chloroadenosine buildingblock (^(Cl) A) was carried out on a synthesis scale of 1 μmol using thecommercially available 5'-O-(4,4-dimethoxytrityl)-N⁶-benzoyl-2'-deoxy-adenosine-3'-phosphonate and the phosphonate fromexample 10 according to a standard protocol (User Bulletin 47, 1988) ona DNA synthesizer 380 B from the Applied Biosystems Company.

The oligomer was processed and purified as described in example 13.

Yield: 3 A₂₆₀ units=ca. 25% of theoretical yield.

EXAMPLE 16

Photochemical conversion of the oligonucleotide from example 15

2.0 A₂₆₀ units of the oligomer from example 14 were dissolved in 0.1 mlredistilled water and irradiated with a 4 W mercury resonance lamp(Heraeus) with a light intensity of 1.79×10¹⁷ quanta×min⁻¹ ×cm⁻². Analmost quantitative conversion into the oligomer containing2'-deoxy-isoguanosine was achieved after 45 minutes according to theHPLC result.

The product proved to be identical when co-injected with the oligomer ofexample 14.

EXAMPLE 172-Bromo-9-(2'-deoxy-β-D-erythro-pentofuranosyl)-6-{[dimethylamino)-methylidene]amino}-9H-purine

The compound was produced analogously to example 8 except that thefollowing amounts were used: 2-bromo-2'-deoxyadenosine (330 mg, 1.0mmol), DMF (3 ml), dimethylformamide-diethylacetal (0.85 ml, 5 mmol).Colourless amorphous solid (306 mg, 80%). TLC (CH₂ Cl₂ /MeOH, 9:1):R_(f) 0.33. UV (MeOH): 318 (28600), 238 (13000). ¹ H-NMR ((D₆ (DMSO):2.32, 2.67 (2 m, H-C(2')); 3.14, 3.23 (2 s, CH₃ -N); 3.56 (m, H-C(5'));3.86 (m, H--C (4')); 4.39 (m, H--C(3')); 4.95 (t, J=5.5 Hz, HO--C (5'));5.33 (d, J=4.3 Hz, HO--C(3')); 6.31 ("t", J=6.3 Hz, H--C(1')); 8.44 (s,H--C(8)); 8.85 (s, H--C(N═)). Anal. calc. for C₁₃ H₁₇ BrN₆ O₃ (385.22):C, 40.53; H, 4.45; N, 21.82; found: C, 40.66; H, 4.50; N, 21.79.

EXAMPLE 182-Bromo-9-[2'-deoxy-5'-O-(4,4'-dimethoxytrityl)]-β-D-erythro-pentofuranosyl)-6-{[(dimethylamino)methylidene]-amino}-9H-purine

The compound was produced as described in example 9. The following wereused: compound from example 17 (250 mg, 0.65 mmol), pyridine (2 ml),4,4'-dimethoxytrityl chloride (330 mg, 0.98 mmol). FC cf. 9. Colourlesssolid (260 mg, 58%). TLC (CH₂ Cl₂ /MeOH, 9:1): R_(f) 0.37. ¹ H-NMR((D₆)DMSO): 2.23, 2.68 (2 m, H--C(2')); 3.01, 3.09 (2 s, CH₃ -N); 3.57,3.58 (2 s, CH₃ O); 3.84 (m, H--C(4')); 4.33 (m, H--C(3')); 5.24 (d,J=4.5 Hz, HO--C(3')); 6.22 ("t", J=5.8 Hz, H--C(1')); 6.6-7.2 (m,aromat. H); 8.21 (s, H--C(8)); 8.69 (s, H--C(N═)). Anal. calc. for C₃₄H₃₅ BrN₆ O₅ (687.59): C, 59.39; H, 5.13; N, 12.22; found: C, 59.46; H,5.40; N, 12.09.

EXAMPLE 199-[2'-Deoxy-5'-O-(4,4'-dimethoxytrityl)-β-D-erythro-pentofuranosyl]-6-[(dimethylamino)methylidene]-9H-isoguanine3'-triethylammonium phosphonate

1,2,4-Triazole (716 mg, 10 mmol) is added to a solution of PCl₃ (270 μl,3.1 mmol) and N-methylmorpholine (3.5 ml) in CH₂ Cl₂ (25 ml). Afterstirring for 30 minutes it is cooled to 0° C. and the compound ofexample 11 (413 mg, 0.64 mmol) dissolved in CH₂ Cl₂ (25 ml) is slowlyadded. After stirring for 30 minutes at room temperature, the mixture isadded to 1 mol/l (Et₃ NH)HCO₃ (35 ml), shaken and the phases areseparated. The aqueous phase is extracted with CH₂ Cl₂ (3×30 ml). Thecombined organic extracts are dried and the colourless foam is separatedon four preparative silica gel plates (20×20 cm) and developed in CH₂Cl₂ -MeOH-triethylamine 80:10:5. The residue from the main zone yields acolourless foam (320 mg, 63%). TLC (CH₂ Cl₂ /MeOH/Et₃ N, 80:10:5): R_(f)0.40. ¹ H-NMR (D₆)DMSO): ¹ H-NMR ((D₆)DMSO): 1.1-1.2 (m, CH₃ CH₂ -N);2.25, 2.65 (2 m, H--C(2')); 2.7-2.8 (m, CH₃ CH₂ N); 3.16, 3.25 (2 s, CH₃-N); 3.25 (s, H--C (5')); 3.77, 3.78 (2 s, CH₃ O); 4.16 (m, H--C(4'));4.79 (m, H--C(3')); 6.20 ("t", J=6 Hz, HO--C(5')); 6.8-7.4 (m, aromat.H); 7.97 (s, H--C(8)); 9.15 (s, H--C(N═)). ³¹ P-NMR ((D₆)DMSO): 2.23 (¹J(PH)=585 Hz, ³ J(PH)=8.1 Hz). Anal. calc. for C₄₀ H₅₂ N₇ O₈ P (789.87):C, 60.82; H, 6.63; N, 12.41; found: C, 60.80; H, 6.79; N, 12.41.

EXMAPLE 202-Bromo-9-[2'-deoxy-5'-O-(4,4'-dimethoxytrityl)]-β-D-erythro-pentofuranosyl)-6-{[dimethylamino)methylidene]-amino}-9H-purine3'-triethylammonium phosphonate

The phosphonate is produced analogously to example 19 from the compoundaccording to example 18. Colourless foam (128 mg, 48%). TLC (CH₂ Cl₂/MeOH/N(Et)₃, 88:10:2): R_(f) 0.35. ¹ H-NMR ((D₆)DMS0): 1.0-1.2 (m, CH₃CH₂ -N); 2.23, 2.82 (2 m, H--C(2')); 2.7-2.9 (m, CH₃ CH₂ N), 3.14, 3.23(2 s, CH₃ -(N═)); 3.70, 3.71 (2 s, CH₃ O); 4.14 (m, H--C(4')); 4.72 (m,H--C(3')); 6.31 ("t", J=6.2 Hz, H--C(1')); 6.63 (d, H--P, J=585 Hz);6.7-7.3 (m, aromat. H); 8.31 (s, H--C(8)); 8.82 (s, H--C(N═)). ³¹ P-NMR((D₆) DMSO): 0.90 (¹ J(PH)=585 Hz, ³ J(PH)=9.1 Hz).

EXAMPLE 212-Chloro-9-(2'-deoxy-β-D-erythro-pentofuranosyl)-6-methylamino-9H-purine

A suspension of 3',5'-bis-toluoyl-2,6-dichloropurine-riboside (A) (1.08g, 2 mmol) in CH₃ N/MeOH (1:5, 30 ml) is stirred for two days at roomtemperature. The reaction mixture is evaporated to dryness in a vacuumand the residue is chromatographed on a silica gel column (2×25 cm, CH₂Cl₂ -MeOH (9:1)). Colourless needles are obtained from water (430 mg,72%). M.p. 163-165° C. TLC (CH₂ Cl₂ -MeOH, 9:1): R_(f) 0.35. UV (water):270 (16800). ¹ H-NMR ((D₆)DMSO): 2.30, 2.65 (2m, H₂ --C(2')); 2.92 (d,J=4.1 Hz, CH₃ -N), 3.55 (m, H₂ -C(5')); 3.86 (m, H--(C-4')); 4.39 (m,H--(C-3')); 4.96 (t, J=5.3 Hz, OH--C(5')); 5.32 (d, J=4.0 Hz,OH--C(3')); 6.27 ("t", J=6.8 Hz, H--C(1')); 8.26 (d, J=4.2 Hz, NH); 8.35(s, H--C(8). Anal. calc. for C₁₁ H₁₄ ClN₅ O₃ (299.7): C, 44.08; H, 4.71;N, 23.27; found: C, 43.96; H, 4.90; N, 23.24.

EXAMPLE 222-Chloro-9-(2'-deoxy-β-D-erythro-pentofuranosyl)-6-hydroxyethylamino-9H-purine

Hydroxyethylamine (915 mg, 15 mmol) is reacted with (A) (810 mg, 1.5mmol) in MeOH (25 ml) as described in example 21. Colourless needles(310 mg, 63%) from EtOAc/acetone. M.p. 167-168° C. TLC (CH₂ Cl₂ -MeOH,9:1): R_(f) 0.15. UV (water): 271 (18700). ¹ H-NMR ((D₆)DMSO): 2.30 and2.65 (2 m, H--C(2')); 3.56 (m, H--C(5'), H--CH₂ (N)); 3.86 (q,H--C(4')); 4.39 (bs, H--C(3')); 4.76 (t, J=4.6 Hz, OH--C(ethyl)); 4.95(t, J=5.5 Hz, OH--C(5')); 5.30 (d, J=4.1 Hz, OH--C(3')); 6.27 (t, J=6.7Hz, H--C(1')); 8.13 (bs, H--N(6)); 8.35 (s, H--C(8)). Anal. calc. forC₁₂ H₁₆ ClN₅ O₄ (329.7): C, 43.71; H, 4.89; N, 21.24; found: C, 43.59;H, 4.80; N, 21.09.

EXAMPLE 232-Chloro-6-butylamino-9-(2'-deoxy-β-D-erythro-pentofuranosyl)-9H-purine

Compound (A) (540 mg, 1 mmol) in MeOH (15 ml) is reacted at 37° C. withN-butylamine (730 mg, 10 mmol) as described in example 21. It ispurified on 20×20 cm preparative silica gel plates using CH₂ Cl₂ -MeOH(9:1) as the mobile solvent. A colourless solid is obtained (210 mg,61%) which crystallizes as needles (water). M.p. 159° C. TLC (CH₂ Cl₂-MeOH, 9:1): R_(f) 0.50. UV (water): 271 (18800). ¹ H-NMR ((D₆)DMSO):0.88, 1.31, 1.56, 3.40 (m, aliphat. H); 2.27 and 2.63 (2 m, H--C(2'));3.56 (m, H--C(5')); 3.86 (bs, H--C(4')); 4.40 (bs, H--C(3')); 4.95 (t,J=5.3 Hz, OH--C(5')); 5.30 (d, J=3.8 Hz, OH--C(3')); 6.27 ("t", J=6.7Hz, H--C(1')); 8.30 (bs, H--N(6)); 8.34 (s, H--C(8). Anal. calc. for C₁₄H₂₀ ClN₅ O₃ (341.8): C, 49.20; H, 5.90; N, 24.49; found: C, 49.16; H,5.85; N, 24.30.

EXAMPLE 242-Chloro-6-cyclohexylamino-9-(2'-deoxy-β-D-erythro-pentofuranosyl)-9H-purine

Cyclohexylamine (1.0 g, 10 mmol) is added to a stirred solution ofcompound (A) (1.08 g, 2 mmol) in MeOH (30 ml). It is stirred for afurther two days at room temperature. The reaction mixture is reactedwith sodium methoxide (3 ml, 1 mol/l in MeOH) and stirred for a furtherday. The reaction mixture is evaporated in a vacuum (oil) and purifiedchromatographically on a silica gel column (2.5×25 cm, using CH₃ Cl-MeOH(9:1) as the mobile solvent). The fractions containing nucleoside areevaporated to dryness in a vacuum. The residue yields colourlesscrystals from EtOAc. (460 mg, 63%). M.p. 160-162° C. TLC (CH₂ Cl₂-MeOH), 9:1): R_(f) 0.63. UV (water): 273 (19600). ¹ H-NMR ((D₆)DMSO):1.014 2.0 (m, H - (cyclohexyl)); 2.25 and 2.60 (2 m, H--C(2')); 3.55 (m,H--C(5')); 3.86 (q, H--C(4')); 4.38 (bs, H--C(3')); 4.97 (t, J=5.6 Hz,OH--C(3')); 5.32 (d, J=4.2 Hz, OH--C (5')); 6.26 ("t", J=6.6 Hz,H--C(1')); 8.15 (d, J=8.4 Hz, H--N(6)); 8.34 (s, H--C(8)). Anal. calc.for C₁₆ H₂₂ ClN₅ O₃ (367.8): C, 52.24; H, 6.03; N, 19.04; found: C,52.11; H, 5.96; N, 18.88.

EXAMPLE 252-Chloro-6-benzylamino-(2-deoxy-β-D-erythro-pentofuranosyl)-9H-purine

Benzylamine (1.07 g, 10 mmol) is reacted with compound (A) (920 mg, 1.7mmol) in MeOH (30 ml) as described in example 24. It is purified bychromatography on a silica gel column (3×24 cm, EtOAc (200 ml) andEtOAc-MeOH (9:1) as the mobile solvent). Colourless crystals areobtained from EtOH/water (390 mg, 61%). M.p. 147° C. TLC (CH₂ Cl₂ -MeOH,9:1): R_(f) =0.60. UV (water): 273 (21600). ¹ H-NMR ((D₆)DMSO): 2.30 and2.60 (2 m, H--C(2')); 3.53 (m, H--C(5')); 3.85 (q, H--C(4')); 4.39 (bs,H--C(3')); 4.64 (d, J=5.7 Hz, H--(CH₂)); 4.96 (t, J=5.9 Hz, OH--C(5'));5.33 (d, J=4.2 Hz, OH--C(3')); 6.27 ("t", J=6.6 Hz, H--C(1')); 7.25-7.32H--C(phenyl)); 8.37 (s, H--C(8)); 8.89 (t, J=4.8 Hz, H--N(6)); Anal.calc. for C₁₇ H₁₈ ClN₅ O₃ (375.8): C, 54.33; H, 4.83; N, 18.64; found:C, 54.27; H, 4.85; N, 18.54.

EXAMPLE 262-Chloro-9-[2'-deoxy-β-D-erythro-pentofuranosyl)-6-methoxy-9H-purine

A suspension of compound (A) (810 mg, 1.5 mmol) in MeOH (30 ml) istreated with sodium methoxide/MeOH (4.5 ml, 1 mol/l) and stirred for 3hours at room temperature. After 30 minutes a clear solution forms. Thereaction mixture is evaporated and the residue is eluted on a silica gelcolumn 60 H (3×22 cm using EtOAc (200 ml) and EtOAc-MeOH (9:1) as themobile solvent). The fractions containing nucleoside are pooled,evaporated in a vacuum and the residue is crystallized from EtOAc.Colourless crystals are obtained (290 mg, 64%). M.p. 141-142° C. TLC(CH₂ Cl₂ -MeOH, 9:1): R_(f) =0.54. UV (water): 258 (12300). ¹ H-NMR((D₆)DMSO): 2.35 and 2.70 (2 m, H--C(2')); 3.56 (m, H--C(5')); 3.85 (q,H--C(4')); 4.10 (s, H--C(6-OCH₃)); 4.42 (bs, H--C(3')); 4.94 (t, J=5.5Hz, OH--C(5')); 5.36 (d, J=4.3 Hz, OH--C(3')); 6.35 ("t", J=6.6 Hz,H--C(1')); 8.60 (s, H--C(8)). Anal. calc. for C₁₁ H₁₃ ClN₄ O (300.7): C,43.94; H, 4.36; N, 18.63; found: C, 44.08; H, 4.45; N, 18.63.

EXAMPLE 276-Amino-2-chloro-9-(2'-deoxy-3',5'-di-O-acetyl-β-D-erythro-pentofuranosyl)-9H-purine

A stirred suspension of 2-chloro-2'-deoxyadenosine (850 mg, 3 mmol) inpyridine (10 ml) is treated with acetic anhydride (10 ml) during whichthe starting material dissolves within 30 minutes. After 3 hours, themixture is evaporated in a vacuum (oil), taken up three times intoluene/EtOH and evaporated. The oily residue is dried in a high vacuumand crystallized from EtOH. (1.03 g, 92%). M.p. 173-175° C. TLC (CH₂ Cl₂-MeOH, 95:5): R_(f) =0.52. UV (MeOH/H₂ O): 264 (15300). 1H-NMR((D₆)DMSO): 1.82 and 2.01 (2 s, H-(CH₂ CO)); 2.55 and 3.00 (2 m,H--C(2')); 4.25 (m, H--C(4') and H--C(5')); 5.37 (m, H--C(3')); 6.29("t", J=6.5 Hz, H--C(1')); 7.85 (s, NH₂); 8.37 (s, H--C(8)). Anal. calc.for C₁₄ H₁₆ ClN₅ O₅ (369.8): C, 45.48; H, 4.06; N, 18.94; found: C,45.61; H, 4.12; N, 18.90.

EXAMPLE 286-Amino-8-bromo-2-chloro-9-(2'-deoxy-3',5'-di-O-acetyl-β-D-erythro-pentofuranosyl)-9H-purine

A solution of 3,5-diacetyl-2-chloro-2-deoxyadenosine (400 mg, 1.08 mmol)in dioxane (16 ml) and aqueous sodium acetate (pH 4.7, 0.5 M, 4 ml) isstirred and a solution of Br₂ (240 mg, 1.5 mmol) in dioxane is addedwithin 15 minutes. It is stirred for a further 15 minutes (monitored byTLC). The mixture is diluted with CHCl₃ (50 ml) and with water (50 ml),sodium bicarbonate (50 ml, sat.), 1% Na₂ S₂ O₄ (50 ml) and water (2×50ml). The organic phase is dried over Na₂ SO₄ and evaporated to dryness.The residue is crystallized from EtOH. Colourless crystals of (8) areformed. (370 mg, 76%). M.p. 163-164° C. TLC (CH₂ Cl₂ -MeOH, 95:5): R_(f)=0.65. UV (MeOH/H₂ O, 1:1): 269 (17500). ¹ H-NMR ((D₆)DMSO): 1.95 and2.09 (2 s, H--C(CH₂ CO); 2.55 and 3.45 (2 m, H--C(2')); 4.17 (m,H--C(5')); 4.34 (m, H--C(4')); 5.33 (q, H--C(3')); 6.29 ("t", J=6.8 Hz,H--C(1')); 7.96 (s, NH₂). Anal. calc. for C₁₄ H₁₅ BrClN₅ O₅ (448.7): C,37.48; H, 3.37; N, 15.61; found: C, 37.63; H, 3.43; N 15.66.

EXAMPLE 296-Amino-8-bromo-2-chloro-9-(2'-deoxy-β-D-erythro-pentofuranosyl-9H-purine(8-bromo-2-chloro-2'-deoxyadenosine)

Ammonia in methanol (10 ml, saturated at 0° C.) is added to a solutionof the compound from example 28 (300 mg, 0.67 mmol) in MeOH (10 ml). Thereaction mixture is stirred at 4° C. overnight. Light-yellowchromatographically-pure crystals are formed (192 mg, 79%). Ananalytical sample is crystallized from ethanol. 190° C. (decomp.). TLC(CH₂ Cl₂ -MeOH, 9:1): R_(f) =0.57. UV (water): 269 (16300). 1H-NMR((D₆)DMSO): 2.20 and 3.15 (2 m, H--C(2')); 3.45 and 3.62 (2 m,H--C(5')); 3.82 (q, H--C(4')); 4.45 (bs, H--C(3')); 4.85 (t, J=6.2 Hz,OH--C(5')); 5.35 (d, J=4.2 Hz, OH--C(3')); 6.23 ("t", J=7.1 Hz,H--C(1')); 7.99 (s, NH₂). Anal. calc. for C₁₀ H₁₁ BrClN₅ O₃ (364.6): C,32.94; H, 3.04; N, 19.21; found: C, 33.12; H, 3.11; N, 19.22.

EXAMPLE 306-[(Dimethylamino)methylidene]-9-(β-D-ribofuranosyl)-9H-isoguanosine

1000 mg (3.53 mmol) isoguanosine is dissolved in 50 ml absolute DMF, 10ml (58.7 mmol) N,N-dimethylformamide-diethylacetal is added and it isstirred for 12 hours at room temperature. Subsequently the solvent isremoved in a vacuum and the residue is evaporated several times firstlywith toluene then with acetone. The residue is purified by columnchromatography (silica gel, column: 25×4.5 cm, mobile solvent CH₂ Cl₂/MeOH 6:4). The residue of the main zone is crystallized from methanol.1054 mg (88.3%) colourless, amorphous crystals which melt above 230° C.while decomposing. TLC (CH₂ Cl₂ /MeOH 3:2): R_(f) 0.45. UV (MeOH: max340, 238 nm. 4.8 (m, H--C(3')); 4.53 (m, H--C(2')); 5.11 (d, HO--C(3'));5.41 (d, HO--C(2')); 5.59 (t, HO--C(5')); 5.65 (d, H--C(1')); 8.05 (s,H--C(8)); 9.16 (s, H--C(N═)); 11.12 (br, s, H--N).

C₁₃ H₁₈ N₆ O₅ (338.33): calc. C, 46.15; H, 5.36; N, 24.84; found C,46.42; H, 5.47; N, 24.75

EXAMPLE 316-[(Dimethylamino)methylidene]-9-[5'-O-(4-methoxytriphenyl-methyl)-β-D-ribofuranosyl]-9H-isoguanosine

150 mg (0.44 mmol) of the compound from example 30 is dried byevaporating twice with absolute pyridine and it is dissolved in 25 mlabsolute pyridine while heating.

206 mg (0.67 mmol) monomethoxytrityl chloride and 227 mg (1.76 mmol)N-ethyldiisopropylamine are added to the hot solution. The reactionmixture is stirred for 12 hours at 60° C. and subsequently 5 ml methanolis added. It is shaken out with 20 ml 5 percent NaHCO₃ solution andextracted 5 times with 10 ml CH₂ Cl₂ in each case. The combined extractsare concentrated and chromatographed on silica gel (column: 15×1.5 cm,mobile solvent: CH₂ Cl₂ /MeOH 83:17). 136.4 mg (50.4%) colourlessamorphous substance. TLC (CH₂ Cl₂ /MeOH, 83:17): R_(f) 0.50. ¹ H-NMR (D₆-DMSO): 3.08, 3.16 (2 s, CH₃ -N); 3.72 (s, CH₃ O); 4.00 (m, H--C(4'));4.16 (m, H--C(3')); 4.49 (m, H--C(2')); 5.16 (d, HO--C(3')); 5.56 (d,HO--C(2')); 5.73 (d, H--C(1')); 6.64 (d, H--C(5')); 7.20-7.37 (m, 14arom. H); 7.96 (s, H--C(8); 9.13 (s, H--C(N═)); 11.11 (br, s, NH).

CH₃₃ H₃₄ N₆ O₆ : calc. C, 64.90; H, 5.61; N, 13.76; found

EXAMPLE 32 2-Amino- (2-deoxy-β-D-erythropentofuranosyl)adenine

5.0 g (18.6 mmol) deoxyguanosine is silylated for 10 hours at 145° C.with 200 ml hexamethyldisilazane (HMDS) and 0.5 ml trimethylchlorosilane(TCS) in the same manner as the corresponding ribo compound [2][3].Subsequently excess HMDS is removed by distillation under normalpressure. The remaining turbid syrup ([2]) is taken up in a mixture of30 ml absolute toluene and 2 ml HMDS. This mixture is transferred into a300 ml autoclave. After addition of 4 ml of a 0.5 M solution (2 mmol) oftris(trimethyl)silyltriflate in absolute toluene, it is pressurized for0.5 hours with NH₃ (5 bar) at 0° C.; then it is heated for 48 hours at145° C. (external temperature regulation). After cooling to roomtemperature, the NH₃ is carefully vented. The solid residue is suspendedin 150 ml methanol (ultrasonic bath), admixed with 150 ml water andtranssilylated for 4 hours at 100° C. After removing the methanol bydistillation, 250 ml water is added, the solution is admixed with activecharcoal, hot-filtered and rewashed with 100 ml hot water. The yellowfiltrate is applied to a Dowex 1×2 column (20×2 cm, OH⁻). Elution with500 ml water yields a main zone from which the product (1.2 g, 24.2%) isobtained after evaporation as a yellow powder. The product is identicalwith an authentic sample [1][4-6). TLC (silica gel, CH₂ Cl₂ /MeOH 4:1):R_(f) =0.4. UV (MeOH):=λ_(max) 217 (23500), 256 (8900), 282 (9900). ¹³C-NMR in ((D₆)DMSO): 160.2 C(6); 156.3 C(2); 151.3 C(4); 135.9 C(8);113.5 C(5); 87.8 C(4'); 83.2 C(1'); 71.1 C(3'); 62.1 C(5').

EXAMPLE 336-Amino-9-(2'-deoxy-β-D-erythro-pentofuranosyl)-1,9-dihydro-2H-purin-2-one(2'-deoxyisoguanosine)

A solution of 300 mg (4.3 mmol) sodium nitrite in 5 ml hot H₂ O isadmixed with 300 mg (1.1 nmol) of the compound from example 32analogously to the ribonucleoside [7] and a total of 0.45 ml (7.8 mmol)glacial acetic acid is slowly added dropwise (foaming!). After 5 minutesit is diluted with 10 ml H₂ O and adjusted to pH 8 with dilute ammoniaand then a further 300 ml H₂ O is added. It is adsorbed to a SerdolitAD-4 ion exchanger (Serva Germany, 4×22 cm) and washed with 500 ml H₂ O.A mixture of about 500 ml H₂ O/i-PrOH (95:5) elutes the main zone fromwhich the compound is obtained as a slightly yellow powder (120 mg,40.8%) after evaporation. TLC (silica gel, H₂ O-saturated n-butanol):R_(f) 0.25. UV (MeOH): λ_(max) 248 (8100), 297 (9900). ¹³ C-NMR in((D₆)DMSO): 156.4 C(6); 137.7 C(8); 153.0 C(2); 109.8 C(5); 88.1 C(4');83.9 C(1'); 71.2 C(3'); 62.0 C(5')[8].

EXAMPLE 34 Silylation of6-[(dimethylamino)methylidene]-9-5'-O-(4-methoxytriphenylmethyl)β-D-ribofuranosyl]-9H-isoguanosinewith triisopropylsilyl chloride

150 mg (0.24 mmol)5'-monomethoxytriethyl-N-6-dimethyl-aminomethylidene-isoguanosine isdried by evaporating twice with pyridine, it is dissolved in 2 mlabsolute pyridine and 62 mg (0.36 mmol) silver nitrite is added. Afterthe silver nitrite has dissolved 0.05 ml (0.25 mmol) triisopropylsilylchloride in 5 ml tetrahydrofuran is added under an argon atmosphere. Thereaction mixture is stirred for 12 hours at room temperature under aninert gas atmosphere in the dark. Afterwards 0.025 ml (0.125 mmol)triisopropyl chloride and 20 mg (0.12 mmol) silver nitrite is againadded. After 30 hours it is neutralized with 10 ml NaHCO₃ solution (5%)and extracted four times with 10 ml dichloromethane in each case. Thecombined extracts are dried over sodium sulfate and concentrated. Theresidue is separated chromatographically.2=2'-triisopropylsilyl-5'-monomethoxytrityl-N-6-dimethylaminomethylidene-isoguanosineis obtained from the main zone with an R_(f) =0.55 (silica gel,EtOAc/CH₂ Cl₂, 4:1). The regioisomer is formed in a small amount.

2'-silylisomer: ¹ H-NMR (D₆ -DMSO): 11.05 (br, s, NH); 9.11 (s, N═CH);7.99 (s, H--C(8)); 7.66-6.84 (m, arom. CH); 5.79 (d, H--C(1')); 5.06 (d,HO--C(3')); 4.77 (m, H--C(2')); 4.21 (m, H--C(3')); 4.03 (m, H--C(4'));3.72 (s, OCH₃); ca. 3.25 (m, H--C(5')); 3.19 and 3.09 (2s, 2 N--CH₃);0.96 and 0.91 (m, Si--C(CH₃)₃); 3.25 (m, 5'-CH₂); 3.19, 3.09 (2 s,[N--(CH₃)₂ ]); 0.9 (m, Si[CH--(CH₃)₂ ]).

EXAMPLE 356-Amino-2-chloro-7-(2'-deoxy-β-D-erythro-pentofuranosyl)-purine

A solution of2,6-dichloro-7-(2'-deoxy-3',5'-di(O-(p-toluoyl)-β-D-erythro-pentofuranosyl)-purine(600 mg, 1.11 mmol) in methanolic ammonia (60 ml, saturated at 0° C.) isstirred for 24 hours at 80° C. The mixture is evaporated in a vacuum andthe residue is chromatographed on silica gel 60 H (column: 15×4 cm).Crystallization from MeOH/iso-PrOH affords colourless crystals (210 mg,66.5%) with a melting point of >250° C. UV (MeOH): λ_(max) 276 nm(ε=7200). ¹ H-NNR ((D₆)DMSO) δ=2.30 and 2.40 (m, 2'-H). 3.56 (m, 5'-H);3.92 (m, 4'-H); 4.40 (m, 3'-H); 5.18 (t, J=5.0 Hz, 5'-OH); 5.42 (d,J=4.5 Hz, 3'-OH); 6.31 (pt, J=6.5 Hz, 1'-H); 7.48 (s, NH₂); 8.56 (s,8-H).

C₁₀ H₁₂ N₅ O₃ Cl (285.67) calc. C, 42.05; H, 4.23; N, 24.51; found C,42.08; H, 4.25; N, 24.58

EXAMPLE 36 7- (2 '-Deoxy-β-D-erythro-pentofuranosyl)-7H-isoguanine

1 ml aqueous ammonia solution is added to a solution of the compoundfrom example 35 (44 mg, 0.16 mmol) in water (75 ml). The mixture isirradiated for eight hours in a quartz container with UV light. Thesolution is evaporated in a vacuum and the residue is dissolved in water(50 ml) and chromatographed on a XAD-4 column (3×20 cm). The column iswashed with water (500 ml) and the product is eluted withwater/isopropanol (1:1). The fractions containing nucleotide are pooled,evaporated in a vacuum and crystallized from ethanol.

[1] Sigma Chemie GmbH, 8024 Deisenhofen, Germany.

[2] H. Vorbruggen, K. Krolikiewicz, Liebigs Ann. Chem. 1976, 745

[3] M. J. Robins, F. Hansske, S. E. Bernier, Can. J. Chem. 1981, 59

[4] R. H. Iwamoto, E. M. Acton, L. Goodman, J. Med. Chem. 1963, 6, 684.

[5] J. A. Montgomery, K. Hewson, J. Med. Chem. 1969, 12, 498.

[6] R. Fathi, B. Goswami, Pei--Pei Kung, B. L. Gaffney, R. A. Jones,Tetrahedron Lett. 1990, 31, 319.

[7] J. Davoll, J. Am. Chem. Soc. 1951, 73, 3174.

[8] Z. Kazimierczuk, R. Mertens, W. Kawozynski, F. Seela, Helv. Chim.Acta 1991, 74, 1742.

What is claimed is:
 1. An oligonucleotide capable of forming a parallelduplex structure, which comprises: ##STR1## wherein R₃ is hydrogen or aprotecting group, is hydrogen, halogen, a reporter group, anintercalator group, --NH(CH₂)_(n) NH₂ wherein n is a number from 2 to12, or --R--CH₂ COOH, wherein R is C₁ -C₈ alkylene.
 2. Theoligonucleotide of claim 1, consisting of from 6 to 100 nucleotides. 3.A compound of formula: ##STR2## wherein R₁ is a protecting group; R₂ isa phosphoramidite or an H-phosphonate;R₃ is hydrogen, or a protectinggroup, and Z is hydrogen, halogen, --NH(CH₂)_(n) NH₂ wherein n is anumber from 2 to 12, or --R--CH₂ COOH wherein R is C₁ -C₈ alkylene, areporter group, or an intercalator group.
 4. A process for making anoligonucleotide, comprising contacting a pre-existing oligonucleotidewith a polymerase and a compound of formula: ##STR3## wherein R₁ is P₃O₉ H₄, or an alpha, beta, or gamma thiophosphate of P₃ O₉ H₄,R₂ ishydroxy, a reporter group or an intercalator group, R₃ is hydrogen or aprotecting group, Z is halogen, --NH(CH₂)_(n) NH₂ wherein n is a numberfrom 2 to 12, or --R--CH₂ COOH, wherein R is C₁ -C₈ alkylene, a reportergroup or an intercalator group, to incorporate said compound into saidpre-existing oligonucleotide.